In airway disease exacerbations, the airway acidifies as measured by low pH in exhaled breath condensate in asthma, COPD and bronchiectasis. Clinically, [unreadable]-adrenergic agonists are used to increase cAMP via stimulation of transmembrane adenylyl cyclases (tmACs), with the intent to enhance mucociliary transport and bronchodilation during disease exacerbations. For bronchodilation, inhaled drugs need to cross the airway epithelium. The majority of the currently used bronchodilators have a positive charge at physiological or acidic pH. Thus, these drugs cannot freely diffuse across the airway epithelium to reach their targets. We have shown that the pH-dependent organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in airway epithelia and that they transport cations, including albuterol, into airway epithelial cells in a pH-dependent manner. Uptake of positively charged bronchodilators is decreased in an acidic airway environment, suggesting that their bronchodilatory effects will be reduced. We have also shown that intracellular acidification directly inhibits ciliary beating. As a counter measure, increases in intracellular HCO3- stimulate cilia via a pathway involving cAMP production by a novel, cytosolic (or soluble) adenylyl cyclase (sAC). These new findings suggest a significant role for pHi and HCO3- in the regulation of human airway homeostasis. We hypothesize that pH directly and HCO3-, likely through sAC, play critical roles in regulating airway epithelial cell functions, including uptake of charged cations such as bronchodilators. This hypothesis will be comprehensively tested with three specific aims, including a bioassay that assesses the ability of inhaled albuterol to dilate airway blood vessels in normal human volunteers. Aim 1 will test the hypothesis that cAMP production is accomplished by both tmACs and sAC and thus influenced by changes in HCO3-, pH, Ca2+. Aim 2 will test the hypothesis that pH-dependent cation uptake into airway epithelial cells will make [unreadable]-adrenergic agonists available in higher concentrations on the airway surface during acidification. Aim 3 will test the hypothesis that airway acidification inhibits transepithelial cation uptake, including albuterol, into airway submucosal tissues in human beings. This application thus addresses a novel and clinically relevant regulation mechanism of mucociliary clearance and of drug availability on the epithelial surface as well as in the subepithelial tissue using a translational and state-of-the-art approach. PUBLIC HEALTH RELEVANCE. Airway diseases and specifically their exacerbations pose important health problems in the USA and worldwide. The airway acidifies during disease exacerbations and this change in pH is predicted to decrease uptake of bronchodilators across the airway epithelium to their target (namely smooth muscle) and adversely affect aspects of mucociliary clearance. This application examines mechanisms by which bronchodilators are taken up into the airway mucosa and novel aspects of mucociliary clearance regulation. Improving ways that bronchodilators reach their target during disease exacerbations and preventing mucociliary dysfunction or restoring clearance to normal levels is the ultimate clinical goal. This proposal thus may identify new therapeutic targets that could help to decrease the burden from airway disease exacerbations on individuals and on society.